Method for delivering a peptide to a subject at a modulated rate via  microcapsules of lactic-co-glycolic copolymer containing said peptide

ABSTRACT

Micro-capsules for the slow release of drugs, consisting of a lactic-co-glycolic copolymer to which a plasticiser has been incorporated and which contain a drug of pharmaceutical interested within them.

The present invention relates to a new type of micro-capsule ormicro-bead for the sustained administration of drugs and to a procedurefor their preparation.

A large variety of administration systems have been proposed for drugsthat require administration over a long time period. The strategydescribed in the literature as the most successful is that ofmicro-encapsulation of the drug to administer in a polymer material ofthe biodegradable and biocompatible polyester type, such aspolylactic-co-glycolic (PLGA). There are a large number of bibliographicreferences to this strategy, such as: U.S. Pat. No. 5,445,832; ES2009346; CH 661 206; CH 665 558; ES 2037621; U.S. Pat. No. 4,652,441; ES2020890; U.S. Pat. No. 4,728,721; U.S. Pat. No. 5,330,767; U.S. Pat. No.4,917,893; U.S. Pat. No. 4,652,441; EP 0 145 240; EP 0 2020 065; EP 0190 833, among others for example.

These polymers have the peculiarity that they are degraded slowly withinthe body releasing the drug contained inside, and the products of thisdegradation (lactic acid and glycolic acid) are naturally present withinthe organism.

In the micro-capsules described in the literature of the state of theart it is very hard to achieve a satisfactory modulation of theencapsulated drug release, and to avoid an initial large drug release,as this can only be achieved by changing the composition of the polymer(the ratio of lactic-glycolic acid or the molecular weight thereof),which usually implies making important changes in the procedure for theproduction of the micro-capsules every time a modification in the drugrelease profile is desired.

In the article published by Pitt el al. in the Journal of BiomedicalMaterials Research, Vol. 13, pg 497-507, 1979, it is described thattributyl citrate accelerates the release of drugs, for example,progesterone, in microcapsules of polylactic polymers.

As a fruit of our research, we have surprisingly discovered that theaddition of small amounts of citric acid esters, to the polymerconstituting the micro-capsules, allows a very effective modulation ofthe liberation characteristics of the micro-capsules obtained, withoutthe need to modify the composition of the polymer.

In the present specification the term modulating release frommicrocapsules is understood to mean a reduction in the initial releaseof encapsulated drug and a release of said drug that is almost linear intime. It is both surprising and unexpected, in view of that described byPitt et al. that the incorporation of small amounts of citric acid esterinto the microcapsule preparation of lactic-co-glycolic polymer thatencapsulate a peptide of pharmaceutical interest allows the release ofthe drug to be almost linear and without the presence of sudden initialreleases of the drug.

Therefore, the object of this invention consists of providingpharmaceutical of prepartions micro-capsules of polymers of lactic andglycolic acid plastified with small quantities of citric acid esters andwhich contain peptides.

The present invention also comprises the preparation and use of theaforementioned microcapsules.

The citric acid esters useful for the purposes of the present inventionare those normally used as plasticizers for pharmaceutical polymers,such as triethyl citrate, tributyl citrate and acetyl tributyl citrate.Use of triethyl citrate is preferable.

By peptides of pharmaceutical interest it is understood:

-   -   analogues of LHRH such as tryptoreline, leuprolide, gosereline,        busereline or cetrorelix    -   analogues of somatostatin such as somatostatin or octreotide    -   analogues of human calcitonin such as salmon calcitonin or        carbocalcitonin.

The preparation of the micro-capsules can be carried out following anyof the methods described in the literature such as, for example, thosedescribed in the U.S. Pat. No. 3,773,919. By way of description andwithout limitation thereto, the different procedures for producingmicro-capsules of the invention would be grouped into the followingsections:

a) Method of Coacervation:

A solution of polymer is prepared along with tri-ethyl citrate in asuitable solvent The drug to be encapsulated is suspended in the polymerand plasticiser solution and a non-solvent of the polymer is added toforce deposition of the polymer on the drug crystals. Examples of theseprocedures without using plasticiser can also be found in documents suchas ES 2009346 or EP 052510.

b) Double Emulsion Methods:

The drug to be encapsulated is dissolved in water or in a solution ofsome other co-adjuvant in is emulsified in a solution of the polymer andthe plasticiser in a suitable solvent such as dichloromethane forexample. The resulting emulsion is in turn emulsified in water or in anaqueous solution of an emulsifier such as polyvinylic alcohol. Once thissecond emulsion has been carried out the solvent in which the polymerwas dissolved is eliminated through evaporation or extraction. Theresulting micro-capsules are obtained directly by filtration. Examplesof these procedures that do not use the plasticiser can also be found indocuments such as U.S. Pat. No. 4,652,441.

c) Simple Emulsion Method:

The drug to be encapsulated, the polymer and the plasticiser aredissolved together in a suitable solvent. This solution is emulsified inwater or a solution of an emulsifier such as polyvinyl acid and theorganic solvent eliminated by evaporation or extraction. The resultingmicro-capsules are recovered by filtration. Examples of these proceduresthat do not sue the plasticiser can also be found in documents such asU.S. Pat. No. 5,445,832.

d) Methods of Solvent Evaporation:

The drug to be encapsulated, the polymer and the plasticiser aredissolved together in a suitable solvent. This solution is evaporated todryness and the resulting residue reduced down to a suitable size.Examples of this procedure, although not using the plasticiser, can bealso be found in documents such as GB 2,209,937.

In the present invention, in all cases, the citric acid ester isdeposited along with the polymer, plastifying it and advantageouslymodifying the hydrophobicity, flexibility and coating capacitycharacteristics of the polymer and the release profile of themicro-capsules obtained.

This is reducing the initial release of the encapsulated drug and makingthis release almost linear in time.

The present invention is now described by means of following,non-limiting examples:

EXAMPLE 1 Production of Micro-Capsules, Containing Leuprolide Acetate,which Presents a Drug Release Profile Suitable for one Month

3 g of tri-ethyl citrate and 1.45 g of lactic-co-glycolic polymer(mw=50000 with monomer ratio of 1/1) are dissolved in 50 ml ofdichloromethane. When the polymer is fully dissolved 67 mg of leuprolideacetate are added and then suspended by sonication.

63 g of silicone of 350 cts is added slowly with intensive stirring. Andwhen all the silicone has been added the content of the reactor ispoured onto 2.5 l of n-heptane and stirred for 1 hour.

The micro-capsules are recovered by filtration and dried under vacuumfor 48 hours.

EXAMPLE 2 Production of Micro-Capsules with One-Month Release ContainingOctreotide Acetate

2 g of tri-ethyl citrate and 1.45 g of lactic-co-glycolic polymer (mw=50000 s5 with monomer ratio of 1/1) are dissolved in 50 ml ofdichloromethane. When the polymer is fully dissolved 67 mg of octreotideacetate are added and then suspended by sonication.

70 g of silicone of 350 cts is added slowly with intensive stirring. Andwhen all the silicone has been added the content of the reactor ispoured onto 2.5 1 of n-heptane. 20 and stirred for 1 hour.

The micro-capsules are recovered by filtration and dried under vacuumfor 48 hours.

EXAMPLE 3 Production of Micro-Capsules with a Three-Month ReleaseProfile Containing Triptoreline Acetate

2 g of tri-ethyl citrate and 1.45 g of lactic-co-glycolic polymer(mw=50000 with monomer ratio of 1/1) are dissolved in 50 ml ofdichloromethane. When the polymer is fully dissolved 45 mg oftriptoreline acetate are added and then suspended by sonication.

70 g of silicone of 350 cts is added slowly with intensive stirring. Andwhen all the silicone has been added the content of the reactor ispoured onto 2.5 l of heptane and stirred for 1 hour.

The micro-capsules are recovered by filtration and dried under vacuumfor 48 hours.

EXAMPLE 4 In vitro Determination of the Drug Release by theMicro-Capsules Obtained Material Needed

12 plastic 10-ml tubes with lid.

1 rack for tubes.

Procedure

Approximately 10 mg of micro-capsules containing leuprolide obtainedaccording to example 1 are weighed into 12 10-ml tubes.

To each tube 2 ml of phosphate buffer 1/30 M and pH=7.0 are added.

Each tube is gently shaken to suspend the micro-capsules in the buffer,the tubes are sealed and placed in an oven at 37° C.

Taking samples for the control of the hydrolysis is carried out inaccordance with the following table:

TABLE 1 Taking samples for analysis of leuprolide released. Tube no.Type of analysis Time  1 h 1, 2 Supernatant  3 h 3 Supernatant  6 h 4Supernatant  1 d 5 and 6  Pellet  2 d 7 Pellet  4 d 8 Pellet Point  8 d10  Pellet 11 d 1 and 11 Pellet 14 d 2 Pellet 18 d 3 and 12 Pellet 23 d9 Pellet 29 d 4 and 5  Pellet

The analysis of leuprolide released is carried out by HPLC in thefollowing conditions:

COLUMN: Kromasil C-8; 25×0.45 cm

ELUENT: Acetonitrile/water 30/70+0.05% trifluoracetic acid

FLOW RATE: 1 ml/min

DETECTION: UV 280 nm.

The samples are taken at the times indicated in table 1 and the analysiscarried out by quantifying the peptide released in the supernatant(supernatant analysis) or the residual peptide inside the micro-capsule(pellet analysis) depending on the hydrolysis time, as indicated intable 1.

The result of this analysis is indicated in FIG. 1. In this figure, theresults obtained are compared with a control assay performed withleuprolide microcapsules in which diethyl citrate has not beenincorporated, in accordance with the method of example 1.

1-24. (canceled)
 25. A method for delivering a peptide of pharmaceuticalinterest at a modulated rate to a subject to whom a plurality ofmicrocapsules of lactic-co-glycolic copolymer containing said peptideare administered, said method comprising administering a sufficientamount of said microcapsules containing the peptide to provide atherapeutic effect to said subject, wherein the copolymer incorporates asufficient amount of a citric acid ester derivative selected from thegroup consisting of triethyl citrate, tributyl citrate and acetyltributyl citrate to modulate the peptide release from said microcapsuleswithin the body of said subject by reducing initial release of thepeptide from the microcapsules and producing a release profile of thepeptide from said microcapsules that is substantially linear over time.26. The method according to claim 25, wherein the citric acid esterderivative is tri-ethyl citrate and the amount of said tri-ethyl citrateranges between 0.1% and 60% of the copolymer.
 27. The method accordingto claim 25, wherein a ratio between lactate and glycolate units in thelactic co-glycolic copolymer is between 100:0 and 10:90, both inclusive.28. The method according to claim 25, wherein the peptide is selectedfrom the group consisting of analogs of LHRH, somatostatin, analogs ofsomatostatin and analogs of human calcitonin.
 29. The method accordingto claim 28, wherein the analog of LHRH is selected from the groupconsisting of tryptoreline, leuprolide, gosereline, busereline andcetrorelix.
 30. The method according to claim 28, wherein the analog ofsomatostatin is octreotide.
 31. The method according to claim 28,wherein the analog of human calcitonin is salmon calcitonin orcarbocalcitonin.
 32. A method for delivering a peptide at a modulatedrate to a subject to whom a plurality of microcapsules containing saidpeptide are administered, said method comprising: (a) forming aplurality of microcapsules containing a peptide by (i) dissolving alactic co-glycolic copolymer and a citric acid ester derivative selectedfrom the group consisting of triethyl citrate, tributyl citrate andacetyl tributyl citrate in a solvent in which said lactic co-glycoliccopolymer and said citric acid ester are soluble to form a solution;(ii) adding a peptide of pharmaceutical interest to the solution to forma peptide suspension in said solution; (iii) adding an alkyl derivativeto the solution to produce deposition of the lactic co-glycoliccopolymer and the citric acid ester derivative on the peptide; (iv)adding the suspension obtained in step (ii) to a solution in which saidlactic co-glycolic copolymer and citric acid ester derivative are notsoluble to harden and precipitate microcapsules thus formed; and (v)isolating the microcapsules thus formed, wherein the citric acid esterderivative is added in an amount sufficient to modulate the peptideliberation characteristics of the microcapsules thus formed, and (b)administering a sufficient amount of said microcapsules containing thepeptide to provide a therapeutic effect to said subject, wherein thecitric acid ester derivative modulates release of said peptide from saidmicrocapsules within the body of said subject by reducing initialrelease of the peptide from the microcapsules and producing a releaseprofile of the peptide from said microcapsules that is substantiallylinear over time.
 33. The method according to claim 32, wherein thecitric acid ester derivative is tri-ethyl citrate and the amount of saidtri-ethyl citrate used in step (i) ranges between 0.1% and 60% of thecopolymer.
 34. The method according to claim 32, wherein a ratio betweenlactate and glycolate units in the lactic co-glycolic copolymer isbetween 100:0 and 10:90, both inclusive.
 35. The method according toclaim 32, wherein the peptide is selected from the group consisting ofanalogs of LHRH, somatostatin, analogs of somatostatin and analogs ofhuman calcitonin.
 36. The method according to claim 35, wherein theanalog of LHRH is selected from the group consisting of tryptoreline,leuprolide, gosereline, busereline and cetrorelix.
 37. The methodaccording to claim 35, wherein the analog of somatostatin is octreotide.38. The method according to claim 35, wherein the analog of humancalcitonin is salmon calcitonin or carbocalcitonin.
 39. The methodaccording to claim 32, wherein the alkyl derivative is silicon oil.